The major goal of this project is to gain a quantitative understanding of the forces and mechanisms that control the assembly, structural stability and functional properties of membrane proteins. As such, three major aspects of the interactions between membrane proteins and phospholipid bilayer membranes will be studied in detail using an experimental thermodynamic approach: 1) The mechanisms and energetics of folding, assembly and stabilization of membrane proteins; 2) The role of the lipid moiety and physicochemical environment on the stability and functional energetics of membrane proteins; and, 3) The energetics of protein insertion into membranes. The research program focuses on two well defined membrane systems: a) Cytochrome c oxidase and b) Diphtheria Toxin. Cytochrome c oxidase is a multisubunit enzyme located in the inner mitochondrial membrane. The three largest subunits are synthesized inside the mitochondria and the remaining subunits are imported from the cytoplasm. We are interested in studying the assembly process and structural stability of the enzyme, the role of the phospholipid bilayer matrix in maintaining the structural and functional integrity of the enzyme, the interactions between subunits, the interactions with its substrate cytochrome c, and the interactions with specific lipid components of the inner mitochondrial membrane (e.g. cardiolipin). Diphtheria toxin is composed of two fragments, A (MW=21,000) and B (MW=37,200), joined by a disulfide bridge. The toxin enters the cell by endocytosis, where it is exposed to a pH of approximately 5. The exposure of the toxin to an acidic environment provides the driving force for the membrane insertion and translocation of the toxic A fragment into the cytosol. One of the goals of this project is to understand protein-membrane interactions that result in the penetration of the protein into the bilayer. The goal in this case is to examine the sequence of protein unfolding -->penetration -->translocation -->refolding in vitro and to provide a complete thermodynamic mechanism for this process. This research project utilizes well defined reconstituted systems and a combination of biochemical and biophysical approaches including different types of calorimetry (high sensitivity differential scanning calorimetry, high sensitivity isothermal] titration calorimetry and multifrequency calorimetry), computer assisted quantitative gel electrophoresis, optical spectroscopy, and other spectroscopic techniques.